1. Field of the Invention
The present invention relates to an extracellular potential sensing element and a device for measuring an extracellular potential, which are used for performing simply and quickly the electrophysiological evaluation of a biological sample, such as a cell, using an electrochemical change generated by the biological sample as an index. The present invention relates also to an apparatus for measuring extracellular potential, and a method of measuring extracellular potential by using the same.
2. Background Art
Drug screening has been conducted using the electrical activity of a cell as an index. Conventionally, the electrical activity of a cell is measured by a patch clamp technique or a technique employing a fluorescent pigment, or a light emitting indicator.
In a patch clamp technique, a small portion (hereinafter referred to as “patch”) of cell membrane is attached to a tip portion of a micropipette, and is used to electrically record with a microelectrode probe the ion transport through a single ion channel protein. The patch clamp technique is one of a few-number of cell biological techniques which can be used to investigate the function of a single protein in real time.
In the other technique, a light generated in response to a change in the concentration of a particular ion is monitored employing a fluorescent pigment, or a light emitting indicator, for measuring the electrical activity of a cell (hereinafter referred to as “fluorescence measuring technique”).
The patch clamp technique requires special techniques for preparation, manipulation and the like of a micropipette, and much time for measuring one sample. Therefore, the patch clamp technique is not suitable for screening a large quantity of candidate compounds for a drug at high speed.
The fluorescence measuring technique can screen a large quantity of candidate compounds for a drug at high speed. However, the fluorescence measuring technique requires a step of staining a cell. During measurement, pigments cause high background noise, and the fluorescence intensity decreases with time, resulting in poor signal to noise ratio (S/N).
An alternative method has been disclosed in WO 02/055653 (hereinafter referred to as “patent document 1”); that is a method of measuring extracellular potential (hereinafter referred to as “extracellular potential measuring method”). The extracellular potential measuring method offers data of high quality level comparable to those by patch clamp technique. Furthermore, the extracellular potential measuring method can measure a large quantity of samples at high speed by a simple process, as the fluorescence measuring technique does.
Patent document 1 discloses an extracellular potential measuring device (hereinafter referred to as “device”), which measures extracellular potential or physicochemical change generated by a cell. The device includes at least one well having means for holding a cell provided on a substrate. The well has a sensing means for detecting an electrical signal.
FIG. 40 illustrates the structure of a typified conventional extracellular potential measuring device. Culture solution 110 is in well 103. Subject cell (cell) 105 is captured or held by cell holding section 113 provided on substrate 102. Cell holding section 113 is formed of depression 104, opening section 106 and throughhole 107 in substrate 102. Throughhole 107 is connected to depression 104 via opening section 106. Detective electrode 109, or sensing means, is disposed in throughhole 109, and detective electrode 109 is connected with a signal detection section (not shown) via wire 108.
During the measurement, cell 105 is sucked by a sucking pump (not shown), or the like means, from the throughhole 107 side, so that it is held to be close to depression 104. At the same time, culture solution 110 flows to the throughhole 107 side and makes contact with detective electrode 109. Thus, an electrical signal generated as the result of activity of cell 105 is detected by detective electrode 109 disposed at the throughhole 107 side, with no leakage into culture solution 110 in well 103.
In the measuring method using a conventional extracellular potential measuring device, cell 105 is reacted with drug (not shown). Therefore, drug needs to be injected into culture solution 110. The injection of drug into culture solution 110 inevitably causes a flow of culture solution 110 in the neighborhood of cell 105. If the change in flow with culture solution 110 is substantial in the neighborhood of cell 105, fluctuation arises with culture solution 111 which is making contact with detective electrode 109. The fluctuation arising in culture solution 111 becomes noise to detecting by detective electrode 109.
In measuring the extracellular potential, the noise to detecting by detective electrode 109 is a cause that deteriorates the S/N ratio of a signal detected by detective electrode 109. Noise is caused by fluctuation of culture solution 111 has a low frequency, approximately 100 Hz or lower. While, the change in extracellular potential exhibits DC signal or a signal that changes with a cycle of less than approximately 5 kHz. Namely, the two signals share an overlapping frequency range.
It has been tried to reduce the noise by means of an electrical filter or the like, but it also cuts off the signal of low frequency region close to DC signal. In some cases, depending on the filter characteristics, even a higher frequency signal of 100 Hz is cut off either. Consequently, it is difficult to detect accurately a signal which represents extracellular potential generated by cell 105.